Structural mimics of RGD-binding sites

ABSTRACT

The present invention provides cyclic peptides that recognize the arginine-glycine-aspartic acid (RGD) motif characteristic of many integrin ligands. These cyclic RGD-binding peptides, which comprise the motif (W/P)DD(G/L)(W/L)(W/L/M), have a structure that functionally mimics the RGD-binding site on an integrin. The invention further provides an antibody selectively reactive with a cyclic RGD-binding peptide containing the sequence (W/P)DD(G/L)(W/L)(W/L/M). The invention also provides a method to reduce or inhibit cell attachment to an RGD-containing ligand using a cyclic RGD-binding peptide of the invention.

This invention was made with government support under grants CA62042,CA28896, and Cancer Center Support grant CA30199 awarded by the NationalCancer Institute. The government has certain rights in the invention.

This application is a continuation of application U.S. Ser. No.08/520,535, filed Aug. 28, 1995, U.S. Pat. No. 5,817,750.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of cell adhesion and morespecifically to integrins and their RGD-binding domains.

2. Background Information

Many cell-cell and cell-matrix interactions depend upon the engagementof specific ligands by members of the integrin family of cell-adhesionreceptors. Integrins are heterodimeric transmembrane receptors whoseligand-binding specificity is determined by the combination of α and βsubunits. Of associations between the nine known β subunits and 17 knownα subunits, integrins α₅ β₁, α_(IIb) β₃ and all or most α_(v)-containing integrins, but generally not others, recognize anarginine-glycine-aspartic acid (RGD) motif. Ligands for theseRGD-binding integrins include a variety of extracellular matrix proteinssuch as fibronectin, vitronectin, osteopontin and collagens; plasmaproteins such as fibrinogen and von Willebrand factor; cellularcounter-receptors; the disintegrins; and viral proteins.

Integrins are fundamental to processes of physical adhesion involvingcell-cell or cell-matrix interactions and also can mediate signaltransduction through their cytoplasmic domains. RGD-binding integrinsfunction in biological processes including cell migration indevelopment, wound healing and tissue repair, platelet aggregation andimmune cell recognition. A role for these integrins also is implicatedin a variety of pathologies including thrombosis, osteoporosis, tumorgrowth and metastasis, inflammation and diseases of viral etiology suchas acquired immune deficiency syndrome. The physiological relevance ofintegrins is underscored by the observation that hereditary mutationscan destroy RGD-binding activity and have pathological consequencesresulting in, for example, the bleeding disorder, Glanzmann'sthrombasthenia.

Peptides and protein fragments can be used to modulate the activities ofRGD-binding integrins. One class of peptides that can act as competitorsof RGD-binding activity includes peptides that contain the RGD motif ora functional equivalent of this motif. A second class of peptidesincludes those peptides that bind RGD-containing ligands throughstructures that function similarly to the integrin domain that contactsthe RGD sequence. Peptides that structurally mimic the RGD-binding sitein integrin β subunits, for example, can modulate the activity ofRGD-binding integrins.

Peptides that specifically bind ligands of RGD-binding integrins wouldbe useful for modulating the cell aggregation and cell adhesion thatoccur in various pathological conditions including, for example,thrombosis, osteoporosis, inflammation, metastasis, wound healing andgraft rejection. However, few such peptides have been described. Thus,there is a need for peptides that effectively and selectively modulatethe activity of RGD-binding integrins. The present invention satisfiesthis need by providing novel cyclic peptides having specific RGD-bindingactivity and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides cyclic peptides that recognize thearginine-glycine-aspartic acid (RGD) motif characteristic of manyintegrin ligands. These cyclic RGD-binding peptides, which comprise themotif (W/P)DD(G/L)(W/L)(W/L/M), have a structure that functionallymimics the RGD-binding site on an integrin. The invention furtherprovides an antibody selectively reactive with a cyclic RGD-bindingpeptide containing the sequence (W/P)DD(G/L)(W/L)(W/L/M). The inventionalso provides a method to reduce or inhibit cell attachment to anRGD-containing ligand using a cyclic RGD-binding peptide of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows binding of RGD-displaying phage to peptides from the β3integrin subunit. (a) Synthetic peptides DYPVDIYYLMDLSYSMKDDLWSIQN (SEQ.ID. NO. 23), designated "DD," and DYPVDIYYLMDLSYSMKAALWSIQN (SEQ. ID.NO. 24), designated "AA," were coated at 100 μg/ml onto microtiter wellsin the presence or absence of 1 mM divalent cations. CELRGDGWC-phage(SEQ. ID. NO. 16) were incubated in the presence or absence of EDTA (10mM) or calcium or magnesium (1 mM). Antibodies against M13 phage wereused to quantify the amount of bound phage. Phage displaying anunrelated peptide sequence, CRDPRAODLC (SEQ. ID. NO. 17), were tested ascontrol phage. , Calcium; ▪, magnesium; , no cations; □, control phage.(b) The effect of soluble GRGDSP (SEQ. ID. NO. 15) or CWDDGWLC (SEQ. ID.NO. 1) peptides on the binding of phage to the same integrin peptides asin a was analyzed. The data in a and b represent the mean values fromtriplicate wells with standard error less than 10% of the mean ▪,Magnesium; , CWDDGWLC (SEQ. ID. NO. 1); , GRGDSP (SEQ. ID. NO. 15); □,control phage.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides cyclic peptides that recognize thearginine-glycine-aspartic acid (RGD) motif characteristic of manyintegrin ligands. These cyclic RGD-binding peptides, which comprise themotif (W/P)DD(G/L)(W/L)(W/L/M), have a structure that functionallymimics the RGD-binding site on an integrin. Peptides of the presentinvention are distinguished by RGD-binding activity. The RGD-bindingactivity of the peptides is further characterized as divalent cationindependent. Cyclic peptides that have RGD-binding activity include, forexample, the peptides CWDDGWLC (SEQ. ID. NO. 1) and CWDDLWWLC (SEQ. ID.NO. 2) as well as other peptides having the consensus sequence(W/P)DD(G/L)(W/L)(W/L/M) shown in Table 2 below. As used herein,underlining of peptide sequences indicates that the structure of thepeptide is cyclic.

As used herein, the term "peptide" refers to linear or cyclic orbranched compounds containing amino acids, amino acid equivalents orother non-amino groups, while still retaining the desired functionalactivity of a peptide. Peptide equivalents can differ from conventionalpeptides by the replacement of one or more amino acids with relatedorganic acids such as p-aminobenzoic acid (PABA), amino acid analogs, orthe substitution or modification of side chains or functional groups.Peptide equivalents encompass peptide mimetics or peptidomimetics, whichare organic molecules that retain similar peptide chain pharmacophoregroups as are present in the corresponding peptide. The term "peptide"refers to peptide equivalents as well as peptides.

It is to be understood that limited modifications can be made to apeptide without destroying its biological function. Thus, modificationsof the peptides of the present invention that do not completely destroytheir RGD-binding activity are within the definition of the compoundclaims as such. Modifications can include, for example, additions,deletions, or substitutions of amino acid residues; substitutions ofcompounds that mimic amino acid structure or function; as well as theaddition of chemical moieties such as amino or acetyl groups.

As used herein, the term "cyclic peptide" refers to a peptide having anintramolecular bond between two non-adjacent amino acids. Thecyclization can be effected through a covalent or non-covalent bond.Intramolecular bonds include, but are not limited to, backbone tobackbone, side-chain to backbone and side-chain to side-chain bonds. Apreferred method of cyclizing peptides of the present invention isthrough formation of a disulfide bond between the side-chains of aminoacids X₁ and X₈. Residues capable of forming a disulfide bond includecysteine (Cys), penicillamine (Pen), β,β-pentamethylene cysteine (Pmc),β,β-pentamethylene-β-mercaptopropionic acid (Pmp) and functionalequivalents thereof (see Table 1).

The peptides disclosed herein also can cyclize, for example, via alactam bond, which can utilize a side-chain group of X₈ to form acovalent attachment to the N-terminal amine of X₁. Residues capable offorming a lactam bond include aspartic acid (Asp), glutamic acid (Glu),lysine (Lys), ornithine (Orn), α,β-diaminopropionic acid, γ-amino-adipicacid (Adp) and M-(aminomethyl)benzoic acid (Mamb). In particular, X₈ ina lactam-bonded peptide can be an aspartic acid or glutamic acidresidue. Cyclization additionally can be

                  TABLE 1    ______________________________________    AMINO ACIDS AND AMINO ACID ANALOGS    USEFUL FOR EFFECTIVE CYCLIZATION                  THREE                  LETTER    AMINO ACID*   CODE         TYPE OF BOND    ______________________________________    γ-amino-adipic acid                  Adp          Lactam    Aspartic acid Asp          Lactam    Cysteine      Cys          Disulfide    Glutamic acid Glu          Lactam    Leucine       Leu          Lysinonorleucine    Lysine        Lys          Lactam and                               Lysinonorleucine    M-(aminomethyl)                  Mamb         Lactam    benzoic acid    Ornithine     Orn          Lactam    Penicillamine Pen          Disulfide    α,β-diaminopropionic                  --           Lactam    acid    β,β-pentamethylene                  Pmc          Disulfide    cysteine    β,β-pentamethylene-                  Pmp          Disulfide    β-mercaptopropionic    acid    Tyrosine      Tyr          Dityrosine    ______________________________________     *- includes amino acids and analogs thereof.

effected, for example, through the formation of a lysinonorleucine bondbetween lysine (Lys) and leucine (Leu) residues or a dityrosine bondbetween two tyrosine (Tyr) residues.

As used herein, the term "RGD-binding activity" refers to an interactionwith a ligand containing an arginine-glycine-aspartic acid (RGD)sequence, or a functional or structural equivalent of this sequence,such that the interaction is specific or selective. Thus, naturallyoccurring RGD-binding sites in integrin β subunits, as well asstructural mimics of RGD-binding sites, are characterized by havingRGD-binding activity.

RGD-containing ligands can be proteins, polypeptides or peptides. Thus,RGD-binding activity refers to the binding of RGD-containing proteins,or fragments thereof, such as vitronectin, fibronectin or fibrinogen, aswell as to the binding of RGD-containing peptides or their functionalequivalents. RGD-binding activity is to be distinguished fromnon-specific binding activity, such as non-specific adsorption to asurface or to a peptide unrelated in sequence to thearginine-glycine-aspartic acid motif. Specific or selective binding canreadily be distinguished from non-specific binding by including theappropriate controls in a binding assay. Several methods for determiningRGD-binding activity are described in Example II.

A distinctive characteristic of such RGD-binding activity is that theinteraction between the RGD-binding domain and the RGD-containing ligandcan be disrupted or prevented by addition of specific competitivesequences. For example, the binding of phage displaying peptides havingRGD-binding activity to fibronectin fragments can be blocked by additionof synthetic peptides encoding RGD or by peptides containing anRGD-binding site, such as the peptides CWDDGWLC (SEQ. ID. NO. 1) orCWDDLWWLC (SEQ. ID. NO. 2).

As used herein, the term "RGD-containing ligand" encompasses proteins,polypeptides and peptides having at least one arginine-glycine-asparticacid sequence, or functional equivalent of an arginine-glycine-asparticacid sequence, which can function as a ligand for an integrin typereceptor. Integrin receptors can bind a variety of RGD-containingpeptides (Ruoslahti et al., In Morphoregulatory Molecules (Edelman etal., 1990); Ruoslahti et al., J. Clin. Invest. 87: 1-5 (1991)).

The term RGD-containing ligand encompasses proteins or peptides whichare functional equivalents of RGD-containing ligands. The term"functional equivalent" in reference to an RGD-containing ligand means aligand having the same or similar activity as an RGD-containing ligand.A functional equivalent of an RGD-containing ligand can, for example,compete for binding to the integrin α_(IIb) β₃. RGD-containing ligandsinclude ligands containing amino acid equivalents of arginine, such aslysine or homoarginine (homoArg), in place of arginine. Similarly,RGD-containing ligands may contain amino acid equivalents of glycine oraspartic acid in place of glycine or aspartic acid, respectively.

As used herein, the term "amino acid equivalents" refers to compoundswhich depart from the structure of naturally occurring amino acids, butwhich have substantially the structure of an amino acid, such that theycan be substituted within a peptide or protein which retains itsbiological activity. Thus, for example, amino acid equivalents caninclude amino acids having side chain modifications or substitutions,and also can include related organic acids, amides or the like. Aminoacid equivalents include amino acid mimetics, which are those structureswhich exhibit substantially the same spatial arrangement of functionalgroups as amino acids but do not necessarily have both the α-amino andα-carboxyl groups characteristic of amino acids. The term "amino acid"refers both to amino acids and amino acid equivalents.

Therefore, a peptide comprising, for example, KGD is considered anRGD-containing ligand within the meaning of the present invention. Inaddition, the anti-α_(IIb) β₃ monoclonal antibodies, PAC1, OPG2 andLJ-CP3, which each contain an RYD sequence as a functional equivalent ofRGD, further exemplify RGD-containing ligands.

The present invention provides cyclic peptides having RGD-bindingactivity, comprising the amino acid sequence:

X₁ X₂ DDX₄ X₅ X₇ X₈ (SEQ. ID. NO. 20),

wherein X₁ and X₈ each is an independently selected amino acid; X₂ andX₇ together equal 0 to 4 amino acids, each of which is independentlyselected; X₄ is selected from the group consisting of glycine andleucine; and X₅ is selected from the group consisting of tryptophan andleucine.

Thus, a peptide of the invention (SEQ. ID. NO. 20) contains a cyclicstructure which consists of less than 11 amino acids. A peptide of theinvention contains two aspartic acid residues followed by a glycine orleucine residue followed by a tryptophan or leucine residue. Therefore,a peptide of the invention includes the consensus motif DD(G/L)(W/L).Furthermore, a peptide of the invention is characterized by havingRGD-binding activity, as defined herein. Such peptides are exemplifiedby CWDDGWLC (SEQ. ID. NO. 1), CWDDLWWLC (SEQ. ID. NO. 2) and CWDDGLMC(SEQ. ID. NO. 3).

In another embodiment, the present invention provides a cyclic peptidehaving RGD-binding activity, comprising the amino acid sequence:

X₁ X₂ X₃ DDX₄ X₅ X₆ X₇ X₈ (SEQ. ID. NO. 22),

wherein X₁ and X₈ each is an independently selected amino acid; X₂ andX₇ together equal 0 to 3 amino acids, each of which is independentlyselected; X₃ is selected from the group consisting of tryptophan andproline; X₄ is selected from the group consisting of glycine andleucine; X₅ is selected from the group consisting of tryptophan and

                  TABLE 2    ______________________________________    SELECTION OF PEPTIDES FROM PHAGE    DISPLAY PEPTIDE LIBRARIES THAT BIND TO FIBRONECTIN    TYPE III.sub.10 RGD-CONTAINING FRAGMENTS       ELUTION             PEPTIDE SEQUENCE     NUMBER    STRATEGY             (SEQ. ID. NO.)                                         OF ISOLATES    ______________________________________    RGD      CWDDGWLC   (SEQ. ID. NO. 1)                                  131    ELUTION                                10LC  (SEQ. ID. NO. 2)                                         7LMC   (SEQ. ID. NO. 3)                                         4MC   (SEQ. ID. NO. 4)    EDTA                          CWDDGWLC   (SEQ. ID. NO. 1)                                          21    ELUTION                    CLVWLLVOFY (SEQ. ID. NO. 5)                                            2                                        1GGGIGRV (SEQ. ID. NO. 6)                                          1ORSC  (SEQ. ID. NO. 7)    DIRECT                   CWDDGWLC   (SEQ. ID. NO. 1)                                          45    INFECTION                          CSWDDGWLC  (SEQ. ID. NO. 8)                                         6    OF WELLS               CPDDLWWLC  (SEQ. ID. NO. 9)                                          3                                      2WLGFC   (SEQ. ID. NO. 10)                                       1RIVLGFTC (SEQ. ID. NO. 11)                                  CDYWLGFC   (SEQ. ID. NO. 12)                                         1                                         1WLVC   (SEQ. ID. NO. 13)                                        1LRC    (SEQ. ID. NO.    ______________________________________                                  14)     .sup.a Peptide sequences disp1ayed by phage isolated by different elution     strategies.?     .sup.b The number of phage displaying the same amino acid motif is     indicated in parenthesis.

leucine; and X₆ is selected from the group consisting of tryptophan,leucine and methionine.

Thus, a peptide of the invention (SEQ. ID. NO. 22) contains a cyclicstructure which consists of less than 11 amino acids. A peptide of theinvention contains a tryptophan or proline residue followed by twoaspartic acid residues followed by a glycine or leucine residue followedby a tryptophan or leucine residue followed by a tryptophan, leucine ormethionine residue. Therefore, a peptide of the invention includes theconsensus motif (W/P)DD(G/L)(W/L)(W/L/M). Furthermore, a peptide of theinvention is characterized by having RGD-binding activity, as definedherein. Examples of such peptides include CWDDGWLC (SEQ. ID. NO. 1),CWDDLWWLC (SEQ. ID. NO. 2) and CWDDGLMC (SEQ. ID. NO. 3).

The present invention further provides cyclic peptides with RGD-bindingactivity having one of the following sequences: CWDDGWLC (SEQ. ID. NO.1); CWDDLWWLC (SEQ. ID. NO. 2); CWDDGLMC (SEQ. ID. NO. 3); CWDDGWMC(SEQ. ID. NO. 4); CSWDDGWLC (SEQ. ID. NO. 8); and CPDDLWWLC (SEQ. ID.NO. 9).

The region responsible for RGD-binding activity has been broadly definedwithin the integrin β subunit. Affinity cross-linking of RGD peptideshas suggested that the ligand-binding site in the platelet receptorα_(IIb) β₃ resides proximal to amino acids 109-171 of the β₃ subunit. Anoverlapping region spanning amino acids 61-203 of β₃ also was implicatedin RGD recognition by cross-linking studies with the vitronectinreceptor α_(v) β₃. In each case, the RGD-binding site is adjacent to orcoincident with a site that binds divalent cations, as is discussedfurther below.

A role for β₃ amino acids 109-171 in RGD binding is further supported bythe high degree of conservation of this region among several integrin βsubunits (76% identity at the amino acid level). Within this conservedregion, amino acids 109-127 are particularly highly conserved amongseveral β subunits, and it has therefore been suggested that thisamino-terminal region is critical to the interaction of integrins withtheir RGD-containing ligands. A comparison of the highly conservedsequence of β₃ (SEQ. ID. NO. 25) with the sequences of several other βsubunits is shown in Table 3 (see Example IV).

Additional studies with mutant integrins support a role for theconserved β₃ (109-127) region in recognition of RGD-containing ligands.Within this conserved segment, a naturally occurring β₃ mutation atAsp¹¹⁹ from a thrombasthenic patient and an analogous mutation in β₁disrupt RGD-dependent binding. In addition, RGD-binding is completelyabsent in Asp¹¹⁹, Ser¹²¹ or Ser¹²³ mutants of α_(IIb) β₃, suggestingthat each of these residues is required for integrin binding toRGD-containing ligands. In contrast, mutations at Asp¹²⁶, Asp¹²⁷ orSer¹³⁰ have relatively minor effects on the RGD-binding of α_(IIb) β₃.These results suggest that the amino-terminal portion of the conservedβ₃ sequence 109-127 is important for RGD recognition by nativeintegrins.

A peptide containing residues 118-131 of β₃ retains RGD-binding activitywhen removed from the context of the intact integrin (D'Souza et al.,Cell 79:659-667 (1994)). Furthermore, residues 118-128, alone, may besufficient for RGD-binding, since a peptide corresponding to β₃(118-128) blocks aggregation of activated platelets as well as plateletadhesion to fibrinogen. In particular, the aspartic acid residue atposition 119 of β₃ appears to be required for ligand binding of the β₃(118-128) peptide because a mutation at this position renders thepeptide ineffective at inhibiting platelet aggregation (D'Souza et al.,Cell 79: 659-667 (1994)).

A distinct region of α_(IIb) β₃ also has been implicated in RGD binding.Specifically, a synthetic peptide containing the β₃ sequence from211-222 can inhibit binding of α_(IIb) β₃ to RGD-containing ligands.This study, with those described above, indicate that there are multipleligand contact points in α_(IIb) β₃.

Thus, several lines of evidence suggest the importance of the highlyconserved region spanning amino acids 109-127 of β₃, although somestudies indicate that conserved residues such as Asp¹²⁶ may not beimportant for function. In particular, the amino-terminal segment ofthis conserved region appears critical since residues Asp¹¹⁹, Ser¹²¹ andSer¹²³ are intolerant of mutation.

The present invention is directed to the surprising discovery that smallcyclic peptides, containing only the C-terminal portion of the 109-127region conserved among β subunits, have RGD-binding activity. The shortpeptide motif, (W/P)DD(G/L)(W/L)(W/L/M), which corresponds to aminoacids 126-131 of β₃, is much smaller than previously describedRGD-binding peptides. Unexpectedly, amino acids 119-123, which includeseveral residues suggested to be important for the RGD-binding activityof intact integrins as described above, are not required for RGD bindingactivity.

The peptides of the present invention are characterized by divalentcation-independent RGD-binding activity. As used herein, the term"divalent cation-independent" refers to RGD-binding activity which doesnot require divalent cations. Divalent cations are positively chargedions which have a valence of two, such as Ca⁺² and Mg⁺². Divalentcation-independent binding is to be distinguished from divalentcation-dependent binding, in which there is a requirement for micromolaror higher concentrations of calcium or magnesium, for example, asdescribed in Example IVB.

In intact integrin receptors, ligand-binding function is dependent upona physiological concentration of divalent cations. Furthermore, theinteraction of the RGD-binding domain with its ligand is dependent uponan "activated" integrin conformation. For example, α_(IIb) β₃, which ismaintained in an inactive conformation on resting platelets, undergoes ameasurable conformational change and becomes competent to bind ligandupon treatment with platelet activators such as thrombin. Since theconformation of α_(IIb) β₃ can be modulated by micromolar or higherconcentrations of Ca⁺² or Mg⁺², receptor-bound cations may be requiredto present the ligand recognition pocket in a conformation competent forbinding.

The β₃ peptide 118-131 has been shown to possess both RGD-binding andcation-binding properties, further emphasizing the intimate relationshipbetween ligand and cation binding for integrin function. In addition,this peptide contains a series of conserved oxygenated residuesreminiscent of the Ca⁺² binding motif known as an "EF hand". Theintegral role of the cluster of conserved oxygenated residues Asp¹¹⁹,Ser¹²¹, Ser¹²³, Asp¹²⁶, Asp¹²⁷ and Ser¹³⁰ within this region issupported, for example, by loss of RGD-binding activity in Asp¹¹⁹,Ser¹²¹ or Ser¹²³ mutants. The conservation of the Ca⁺² binding EF handmotif and the demonstrated functional importance of several oxygenatedresidues in β₃ (118-131) implies that a functional calcium-binding motifis required for RGD binding activity.

The present invention is directed to the surprising discovery thatdivalent cations are not required for the binding of the cyclic peptidesof the invention to RGD-containing ligands. As discussed above, a bodyof evidence supports the divalent cation requirement in intactintegrins. Furthermore, the presence of a Ca⁺² binding "EF hand" motifin a peptide with RGD-binding activity also suggested the importance ofdivalent cations for RGD-binding. In contrast with what was believed bythose skilled in the art, the present invention provides novel cyclicpeptides capable of binding to RGD in the absence of divalent cations.

Cyclic peptides of the present invention are useful for identifying andisolating novel integrin ligands in solid phase assays. In such solidphase assays, the cyclic peptides of the invention are immobilized on asolid support. Such peptides, which have divalent cation-independentRGD-binding activity, are preferred for use in solid phase assays ascompared to longer linear RGD-binding peptides, which must first foldand assume a cation binding conformation prior to binding ligand. Such aconformation can be hindered by the solid support. Similarly, the cyclicpeptides of the invention can be useful for identifying novel moleculesthat reduce or inhibit the binding of RGD-containing ligands tointegrins. In such an assay, the binding of cyclic peptides of theinvention and known RGD-containing ligands would be assayed to determinewhether a molecule could reduce or inhibit binding.

Specific cyclic peptides of the present invention can be isolated by avariety of methods based on their RGD-binding activity. For example,peptides characterized by specific RGD-binding activity may beidentified by screening a large collection, or library, of random cyclicpeptides or cyclic peptides of interest. Cyclic peptide librariesinclude, for example, tagged chemical libraries comprising peptides andpeptidomimetic molecules. Cyclic peptide libraries also comprise thosegenerated by phage display technology. Phage display technology includesthe expression of peptide molecules on the surface of phage as well asother methodologies by which a protein ligand is or can be associatedwith its encoding nucleic acid. Methods for the production of phagedisplay libraries, including vectors and methods of diversifying thepopulation of peptides which are expressed, are well known in the art.(See, for example, Smith and Scott, Methods Enzymol. 217: 228-257(1993); Scott and Smith, Science 249: 386-390 (1990); and Huse, WO91/07141 and WO 91/07149, each of which is incorporated herein byreference; see, also, Example I). Cyclic peptide libraries also are wellknown in the art (see, for example, Koivunen et al., Methods Enzymol.245: 346-369 (1994)). These or other well known methods can be used toproduce a phage display library, from which peptides of the inventioncan be isolated using a variety of assays for RGD-binding activity.Other methods for producing RGD-binding cyclic peptides include, forexample, rational design and mutagenesis.

RGD-binding assays are well known in the art. Such well known bindingassays include ELISA and radioreceptor assays. A cyclic peptide librarycan be screened for RGD-binding activity using any one of a number ofRGD-containing ligands. RGD-binding activity can be assayed, forexample, using native RGD-containing proteins such as vitronectin andfibronectin as demonstrated in Example III. Protein fragments retainingat least one RGD sequence, such as the 10^(th) type III repeat offibronectin, or RGD-containing peptides also can be used as ligands toassay RGD-binding activity (see Example I). As discussed above,RGD-containing peptides encompass anti-α_(IIb) β₃ monoclonal antibodiesthat contain an RYD sequence as a functional equivalent of RGD. Suchmonoclonal antibodies, for example PAC1, OPG2 and LJ-CP3 which are wellknown in the art, similarly can be used to screen peptide libraries forRGD-binding peptides. Furthermore, specific RGD-binding can bedistinguished from non-specific binding by the inclusion of appropriatecontrols, such as a non-RGD-containing fibronectin fragment or anunrelated protein such as bovine serum albumin (see Example I).

The peptides of the present invention can be isolated or synthesizedusing methods well known in the art. Such methods include recombinantDNA methods and chemical synthesis. Recombinant methods of producing apeptide through expression of a nucleic acid sequence encoding a peptidein a suitable host cell are well known in the art, such as is describedin Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed, Vols1 to 3, Cold Spring Harbor, N.Y. (1989), which is incorporated herein byreference.

Peptides of the invention can also be produced by chemical synthesis,for example, by the solid phase peptide synthesis of Merrifield(Merrifield et al., J. Am. Chem. Soc., 85:2149 (1964), which isincorporated herein by reference). Standard solution methods well knownin the art also can be used to synthesize a peptide of the presentinvention (see, for example, Bodanszky, M., Principles of PeptideSynthesis (Springer-Verlag, 1984), which is incorporated herein byreference). Newly synthesized peptides can be purified, for example, byhigh performance liquid chromatography (HPLC), and can be characterizedusing, for example, mass spectrometry or amino acid sequence analysis.Methods exemplifying the synthesis and purification of peptides areprovided in Example IIA.

A newly synthesized linear peptide can be cyclized by the formation of abond between reactive amino acid side chains. For example, a peptidecontaining a cysteine-pair, or any of the cysteine analogs shown inTable 1, can be synthesized, and a disulfide bridge can be formed byoxidizing the peptide with 0.01 M K₃ Fe(CN)₆ ! at pH 8.4, as describedin Example IIA. Alternatively, a lactam, a lysinonorleucine or adityrosine bond can be formed. Methods for forming these and other bondsare well known in the art and are based on well established principlesof chemical reactivity (Morrison and Boyd, Organic Chemistry, 6th Ed.(Prentice Hall, 1992), which is incorporated herein by reference).

A peptide of the present invention also can be cyclized by forming apeptide bond between non-adjacent amino acid residues as described, forexample, by Schiller et al., Int. J. Pept. Prot. Res. 25:171 (1985),which is incorporated herein by reference. Peptides can be synthesizedon the Merrifield resin by assembling the linear peptide chain usingN.sup.α -Fmoc-amino acids and Boc and tertiary-butyl proteins. Followingrelease of the peptide from the resin, a peptide bond can be formedbetween the amino and carboxyl termini.

In another embodiment, the present invention provides an antibodyselectively reactive with a cyclic peptide containing the sequence(W/P)DD(G/L)(W/L)(W/L/M). An antibody raised against a cyclicRGD-binding peptide of the invention can be useful for detectingmultiple integrin β subunits in various applications.

As used herein, the term "selectively reactive" refers to an antibodywhich can distinguish, or can be made to distinguish, related sequencesover unrelated sequences. The term related sequences as used hereinincludes identical peptide or protein sequences as well as different butsimilar peptide or protein sequences. Thus, antibodies selectivelyreactive with a peptide of the invention will react with sequences whichinclude, for example, CWDDGWLC (SEQ. ID. NO. 1) and CWDDLWWLC (SEQ. ID.NO. 2). Such antibodies also can react with the integrin β₃ subunit, orrelated β subunits, which contain the sequence DDLW or DDL.

As used herein, the term "antibody" refers to polyclonal and monoclonalantibodies, as well as polypeptide fragments of antibodies that retainselective reactivity for a peptide of the invention. One skilled in theart would know that antibody fragments such as Fab, F(ab')₂ and Fvfragments can retain selective reactivity for cyclic peptides of theinvention and, thus, are included within the definition of an antibody.In addition, the term "antibody" as used herein includes naturallyoccurring antibodies as well as non-naturally occurring antibodies andfragments that retain binding activity. Such non-naturally occurringantibodies can be constructed using solid phase peptide synthesis,produced recombinantly or obtained, for example, by screeningcombinatorial libraries consisting of variable heavy chains and variablelight chains as described by Huse et al., Science 246:1275-1281 (1989),which is incorporated herein by reference.

Particularly useful non-naturally occurring antibodies include chimericantibodies and humanized antibodies. Chimeric antibodies and humanizedantibodies are useful for administration to a human subject, since thelikelihood of an immune response by the subject against the antibody isminimized.

Methods for producing an antibody are routine in the art as described,for example, by Harlow and Lane, Antibodies: A Laboratory Manual (ColdSpring Harbor Laboratory Press, 1988), which is incorporated herein byreference. One skilled in the art would know that a cyclic peptideuseful as an immunogen can be produced recombinantly or can bechemically synthesized, as described in detail above. In some cases, acyclic peptide of the invention can be made more immunogenic by couplingthe hapten to a carrier molecule such as bovine serum albumin or keyholelimpet hemocyanin. In addition, various other carrier molecules andmethods for coupling a hapten to a carrier molecule are well known inthe art (see, for example, Harlow and Lane, supra, 1988).

Polyclonal antibodies selectively reactive with a cyclic peptide of theinvention can be raised in rabbits for example. In addition, monoclonalantibodies can be obtained using known methods (Harlow and Lane, supra,1988). Similarly, methods for identifying an antibody selectivelyreactive with a cyclic peptide of the invention are known in the art andinclude, for example, enzyme-linked immunosorbent assays,radioimmunoassays and precipitin assays (Harlow and Lane, supra, 1988;chap. 14).

In another embodiment, peptides of the present invention are used toreduce or inhibit integrin-mediated cell attachment to an RGD-containingligand by administering an effective amount of the RGD-binding peptide.The RGD-containing ligand can be an extracellular matrix protein, forexample, such as vitronectin, fibronectin, osteopontin or fibrinogen.

As used herein, the term "cell attachment" is meant to include theattachment of cells to other cells and to RGD-containing ligands such asextracellular matrix proteins and certain viruses. Platelet aggregationis an example of cell attachment, as is the attachment of cells toinsoluble substrates such as fibronectin or vitronectin. The term cellattachment encompasses the attachment of cells in vitro and in vivo.Assays to measure cell attachment are well known in the art and aredescribed in Example III.

As used herein, the term "effective amount" means the amount of apeptide useful for reducing or inhibiting cell attachment in vitro or invivo. An effective amount for reducing or inhibiting cell attachment canbe determined using methods known to those in the art, including theassay described in Example III.

Peptides of the present invention are useful in modulating the activityof RGD-binding integrins such as α₅ β₁, α_(IIb) β₃ and α_(v) β₃ due tothe ability of the claimed peptides to reduce or inhibit cell attachmentby binding RGD-containing ligands. Inhibition of cell attachment bypeptides of the invention is useful in the treatment of pathologiesresulting from abnormal integrin-mediated cell attachment, such asthrombosis, osteoporosis, inflammation, tumor growth or metastasis.Unlike currently available RGD-containing peptides, which target thecell carrying the RGD-binding integrin, peptides of the presentinvention target the integrin ligand. Since the binding of anRGD-containing peptide to an integrin has been shown to be capable ofinducing signaling by the integrin, peptides of the present invention,which bind to a different target, add a useful alternative to thecompounds currently available for inhibition of cell attachment.

Thus, cyclic peptides of the invention can reduce or inhibit the bindingof platelets to fibrinogen by administration of a sufficient quantity ofpeptide to the cells. The reduction or inhibition of integrin binding tofibrinogen is useful, for example, in the treatment of thrombosis, sinceα_(IIb) β₃ mediates platelet aggregation. Peptides of the invention alsocan reduce or inhibit infection by several types of viruses that use anRGD-containing protein to gain entry to host cells. Such virusesinclude, for example, hoof-and-mouth viruses and certain adenoviruses.Similarly, the cyclic RGD-binding peptides of the invention can beuseful in inhibiting or reducing the severity of other pathologiesresulting from abnormal integrin-mediated cell attachment.

EXAMPLE I IDENTIFICATION OF RGD-BINDING PEPTIDES

This example describes isolation of peptides of the invention.

A. Isolation of Phage Capable of Binding to Fibronectin Fragments

To isolate peptides that interact with the RGD-containing 10th type IIIdomain of fibronectin (III₁₀), recombinant fibronectin fragments wereused to select clones from a mixture of peptide libraries by successiverounds of affinity panning and elution with RGD-containing peptide.Phage display libraries were made as described (Koivunen et al., MethodsEnzymol. 245:346-369 (1994), which is incorporated herein by reference)using the fuse 5 vector (Smith and Scott, Methods Enzymol. 217: 228-257(1993), which is incorporated herein by reference). Mixtures oflibraries displaying CX₅ C, CX₆ C, CX₇ C and CX₉ (wherein X₅, X₆, X₇ andX₉ represent a sequence of 5, 6, 7 or 9, respectively, randomly selectedamino acids) peptides were screened for binding to fibronectin fragmentscoated on microtiter wells.

The fibronectin fragments used to coat the wells were prepared asfollows. Human plasma fibronectin was from the Finnish Red Cross(Helsinki, Finland). A 110-kD fragment of fibronectin was prepared asdescribed previously in Pierschbacher et al., Cell 26: 259-267 (1981)which is incorporated herein by reference. Recombinant fibronectinfragments containing type III repeats 8 and 9, 9 and 10, 10 and 11, 10alone, and 8 through 11 were produced as described (Dickinson et al., J.Mol. Biol. 238: 123-127 (1994), which is incorporated herein byreference), using the GST protein fusion system (Pharmacia, Uppsala,Sweden) for the 8 through 11 fragment and the His-Tag fusion proteinsystem (Qiagen, Chatsworth, Calif.) for the other four fragments. Afragment encompassing the alternatively spliced cell attachment domainof fibronectin, which comprises amino acids 1860-2140, was also producedusing the His-Tag system.

Panning was performed on each fragment individually. In the first andsecond rounds of panning, the coating concentration of protein was 5μg/well. To increase the stringency of the panning, the wells werecoated with decreasing concentrations of protein: 1.0 μg/well and 0.1μg/well. Phage were selected for further amplification from the wellwith the lowest protein concentration that showed phage binding overbackground, where binding was detected and quantified by infection ofbacteria. In the fourth panning, the concentration of fibronectinfragment was 10 ng/well. To recover the bound phage, the wells wereeluted with a 1 mM solution of GRGDSP (SEQ. ID. NO. 15) or CELRGDGWC(SEQ. ID. NO. 16) peptides, 2 mM EDTA or were directly incubated with 50μl of bacteria. Phage were sequenced from randomly selected clones asdescribed in Koivunen et al., Methods Enzymol. 245: 346-369 (1994).

Decreasing protein coating concentration in the second and third roundsof panning, and the use of an excess of phage to introduce bindingcompetition, allowed for selection of specific phage that bound withhigh-affinity to the fibronectin fragments. In the third round ofpanning, 50- to 150-fold enrichment was achieved on III₁₀ -bearingfragments. The fragment containing the 10^(th) and 11^(th) type IIIrepeats of fibronectin was the most efficient binder of specific phage.Enrichment was also seen on the recombinant fibronectin fragment bearingthe III₁₀ domain alone, but not on the fragment from the alternativelyspliced fibronectin domain containing the CS-1 binding site for β₄ β₁integrin (results not shown). Binding of phage to fragment III₈,9 wasalso low, indicating that the III₁₀ domain was important for theenrichment of RGD-eluted phage. Glutathione S transferase (GST) andbovine serum albumen (BSA) were used as controls for non-specificattachment and showed negligible phage binding. Enrichment of specificphage was also seen with the RGD-coating fragments when the phage wereeluted with EDTA or collected by direct infection of bacteria added tothe washed wells.

B. Phagre Selected by RGD-containing Fibronectin Fragments Display the(W/P)DD(G/L)(W/L)(W/L/M) Peptide Motif

Sequences of the insert in the phage eluted with RGD peptides, EDTA, orrecovered by direct incubation of bacteria showed that approximately80-85% of the clones displayed the motif CWDDGWLC (SEQ. ID. NO. 1) (seeTable 2 above). Furthermore, the CWDDGWLC (SEQ. ID. NO. 1) sequence wasnot encoded by a single clone, since there was variation at thenucleotide level among the phage.

Some of the other motifs found were similar to the CWDDGWLC (SEQ. ID.NO. 1) sequence: the glycine residue in the fifth position wasfrequently replaced by leucine. In addition, the tryptophan in thesecond position could be replaced by proline; the tryptophan in thesixth position could be replaced by leucine; and the leucine in theseventh position could be replaced by tryptophan or methionine.

The sequences that were not related to the (W/P)DD(G/L)(W/L)(W/L/M)motif were hydrophobic and/or were seen only once. The binding of thesephage is likely to have been non-specific. These rarely isolatedpeptides were lost in the subsequent high affinity screening steps andwere not seen at all when specific elution with an RGD peptide was used.

EXAMPLE II ASSAYS FOR RGD-BINDING ACTIVITY

This example describes specific RGD-binding assays for the analysis ofphage displaying peptides and for the analysis of synthetic peptides.

A. Binding of CWDDGWLC Phage to the Fibronectin III₁₀ Domain is Blockedwith Synthetic Peptides

The specificity of the CWDDGWLC-phage (SEQ. ID. NO. 1) binding tofibronectin and fibronectin fragments was tested in a microtiter assay.

The phage attachment assay was performed by binding individual clonedphage to insolubilized fibronectin or fibronectin fragments inmicrotiter assays as described (Koivunen et al., Methods Enzymol. 245:346-369 (1994) and Koivunen et al., J. Cell Biol. 124: 373-380 (1994),which are each incorporated herein by reference). The coatingconcentration for the proteins was 10 μg/ml. Coating with peptides wascarried out at 10-100 μg/ml overnight with or without 1 mM divalentcations. Phage binding was determined by growing K91kan bacteria in thepresence of the selection marker tetracycline. The absorbance at 600 nmwas read after 16-24 hours of incubation at room temperature (Koivunenet al., Methods Enzymol. 245: 346-369 (1994)). Alternatively, phagebinding was quantified using sheep anti-M13 polyclonal antibodies (1μg/ml; Pharmacia). Readings at 450 nm were analyzed after incubationwith alkaline phosphatase conjugated anti-sheep IgG (1:10,000; Sigma).

The binding was dependent on the presence of the III₁₀ domain. Bindingto III₈,9, to control proteins (BSA and GST) and also to the fragmentencompassing the alternatively spliced cell attachment domain offibronectin was minimal. An unrelated phage displaying the peptideCRDPRAODLC (SEQ. ID. NO. 17) showed no binding to fibronectin or any ofits fragments.

Two cyclic peptides, CWDDGWLC (SEQ. ID. NO. 1) and ACRGDGWMCG (SEQ. ID.NO. 18), were synthesized and tested for their ability to inhibit phagebinding to the III₈,11 fragment. Peptides were synthesized on asynthesizer (Model 430A: Applied Biosystems, Foster City, Calif.) bystandard Merrifield solid phase synthesis protocols and t-butoxycarbonylchemistry. Cyclic peptides were prepared by oxidizing with 0.01M K₃Fe(CN)₆ at pH 8.4 overnight and purified by reverse-phase HPLC. Thepeptide structures were confirmed by mass spectroscopy. Both peptides,but not an irrelevant cyclic one (i.e., GACVRLNSLACGA) (SEQ. ID. NO.19), inhibited CWDDGWLC-phage (SEQ. ID. NO. 1) binding in adose-dependent manner. EDTA did not inhibit the binding ofCWDDGWLC-phage (SEQ. ID. NO. 1), although CWDDGWLC-phage (SEQ. ID.NO. 1) were detected in the EDTA eluates after affinity panning. Therecovery of the CWDDGWLC-phage (SEQ. ID. NO. 1) with EDTA, (Table 2),may have represented non-specific release of the bound phage rather thanspecific elution, because fewer phage were eluted with EDTA than withthe RGD peptide.

Therefore, binding of the CWDDGWLC (SEQ. ID. NO. 1) phage to theRGD-containing fibronectin fragments was specific because onlybackground binding was seen when fragments lacking the RGD-containingIII₁₀ domain, or control proteins, were used. Moreover, the specificityof the CWDDGWLC-phage (SEQ. ID. NO. 1) binding to fibronectin fragmentsalso was supported by specific inhibition of the interaction by peptidesrepresenting the motif itself and by RGD-containing peptides.

B. Phage Attachment to Immobilized Peptides

The binding of CWDDGWLC (SEQ. ID. NO. 1) and RGD-displaying phage to RGDand CWDDGWLC-containing (SEQ. ID. NO. 1) peptides was analyzed in phageattachment assays using CWDDGWLC (SEQ. ID. NO. 1), ACRGDGWMCG (SEQ. ID.NO. 18), and an irrelevant cyclic peptide GACVRLNSLACGA (SEQ. ID. NO.19) as substrates. RGD- and CWDDGWLC (SEQ. ID. NO. 1) containing cyclicpeptides were coated on microtiter wells at 20 μg/ml and used to bindphage. Soluble peptides were added at 1 mM concentration. CWDDGWLC-phage(SEQ. ID. NO. 1) bound to immobilized RGD-containing peptide.Conversely, RGD-phage, i.e., phage displaying a peptide containing theCELRGDGWC motif (SEQ. ID. NO. 16), bound to immobilized CWDDGWLC (SEQ.ID. NO. 1). The RGD phage also showed slight, but consistent, binding tothe peptide displaying the same RGD motif, RGDGW. Addition of either theCWDDGWLC (SEQ. ID. NO. 1) or ACRGDGWMCG (SEQ. ID. NO. 18) peptide insolution blocked the CWDDGWLC-phage (SEQ. ID. NO. 1) binding, whereas anunrelated peptide had no effect. The binding of RGD-phage was alsoinhibited by both peptides, but was unaffected by control peptide or byEDTA.

C. Fibronectin Binds to CWDDGWLC-sepharose

Affinity chromatography showed that fibronectin bound toCWDDGWLC-Sepharose (SEQ. ID. NO. 1). Peptides were coupled toSepharose-CH (Pharmacia) according to manufacturer's instructions.Fibronectin or fibronectin fragments at 2 mg/ml in TBS were applied toCWDDGWLC-Sepharose (SEQ. ID. NO. 1). After extensive washing with TBS,bound fibronectin was eluted with the GRGDSP (SEQ. ID. NO. 15) peptide(1 mM) but not with the GRGESP (SEQ. ID. NO. 21) peptide (1 mM), asdetermined by analysis of eluate samples by SDS-PAGE followed byCoomassie blue staining. Fibronectin was not retained in a controlunrelated peptide column. Fibronectin fragments lacking the III₁₀ domain(i.e., fragment III₈,9) and unrelated proteins (BSA and type IVcollagen) were not retained in the CWDDGWLC-Sepharose (SEQ. ID. NO. 1)column, as determined by analysis of fractions eluted in glycine/NaCl(pH 3.0) for protein by measuring OD280.

EXAMPLE III CELL ATTACHMENT ASSAY

This example describes an assay for peptide inhibition of the cellattachment function of integrins.

A. CWDDGWLC Inhibits RGD-dependent Cell Adhesion

CWDDGWLC (SEQ. ID. NO. 1) peptide inhibition of integrin function wasassayed in cell attachment assays with the osteosarcoma cell line, MG-63which attaches to fibronectin, vitronectin, and collagens through itscomplement of several integrins (Pytela et al., Methods Enzymol. 144:475-489 (1987)). Microtiter wells were coated with fibronectin,vitronectin or type IV collagen at concentrations that resulted in 60%of maximal attachment (˜5 μg/ml). Human plasma fibronectin was from theFinnish Red Cross (Helsinki, Finland). Vitronectin was purified fromhuman plasma as described in Yatohgo et al., Cell Struc. Funct. 13:281-292 (1988), which is herein incorporated by reference. Collagen wasfrom Collaborative Research (Bedford, Mass.). Free binding sites onplastic were blocked with BSA. Approximately 1×10⁵ cells per well wereallowed to attach for 30 min in the presence or absence of competingpeptides and the bound cells were quantitated by staining with crystalviolet (Morla et al., Nature 367: 193-196 (1994)).

The CWDDGWLC (SEQ. ID. NO. 1) peptide inhibited cell adhesion wheneither fibronectin or vitronectin were used as substrates, but not oncollagen type IV. An unrelated cyclic peptide had no effect on any ofthe substrates. The peptide CWDDGWLC (SEQ. ID. NO. 1) was slightly lesseffective than the standard RGD peptide, GRGDSP (SEQ. ID. NO. 15).

EXAMPLE IV STRUCTURAL SIMILARITY TO THE β3-INTEGRIN SUBUNIT

This example provides a method for the production of antibodies againstthe CWDDGWLC (SEQ. ID. NO. 1) peptide. This example further describes amethod for analyzing the structural relationship between the cyclicCWDDGWLC (SEQ. ID. NO. 1) peptide and integrin β subunits.

A. *CWDD^(G) /_(L) WLC* Resembles a Peptide from β3 Integrin Subunit

A DDLW sequence from the ligand binding region of the β subunits showssimilarity to the RGD binding peptides of the invention (see Table 3). Asynthetic peptide containing the DDLW sequence of the β3 subunit (aminoacids 109-133; DYPVDIYYLMDLSYSMKDDLWSIQN (SEQ. ID. NO. 23)) has beenshown to bind to an RGD peptide (D'Souza et al., Cell 79: 659-667(1994)). As shown in FIG. 1, RGD-phage bound to the β3(109-133) peptide(SEQ. ID. NO. 23).

                  TABLE 3    ______________________________________    COMPARISON OF THE RGD-BINDING SEQUENCE    FROM PEPTIDE LIBRARY WITH INTEGRIN SEQUENCES                  CWDDG/LWLC    ______________________________________    β3: DIYYLMDLSYSMKDDLWSIQNLGTKLAT  (SEQ. ID. NO 25)    β1: DLYYLMDLSYSMKDDLENVKSLGTDLMN  (SEQ. ID. NO 26)    β5: DLYYLMDLSLSMKDDLDNIRSLGTKLAEE (SEQ. ID. NO 27)    β6: DLYYLMDLSAAMDDDLNTIKELGSGLSKE (SEQ. ID. NO 28)    ______________________________________     The amino acid sequences of integrin β subunits are shown using the     sing1e1etter code. The sequence of β3 that is shown encompasses     residues 113-140. Residues D.sup.119, D.sup.126, and D.sup.127 within the     β.sub.3 subunit are underlined.

The binding required the presence of divalent cations, either while thepeptide was coated onto plastic or during the phage binding (FIG. 1a).The binding of the RGD-phage was blocked by addition of soluble CWDDGWLC(SEQ. ID. NO. 1) or GRGDSP (SEQ. ID. NO. 15) peptides (FIG. 1b). The twopeptides were equally effective in inhibiting RGD-binding to β₃(109-133) (SEQ. ID. NO. 23): In titration experiments, 50% inhibitionwas achieved at about 1 μM concentration of the peptides (not shown).However, EDTA did not block phage binding when peptides were allowed tocoat in the presence of cations.

To establish whether the DDLW sequence in the β₃ (109-133) peptide (SEQ.ID. NO. 23) was important for the RGD-phage binding, we synthesized avariant β₃ peptide in which the aspartate residues at positions 126 and127 were replaced by alanines (DYPVDIYYLMDLSYSMKAALWSIQN (SEQ. ID. NO.24) designated "AA"). RGD-phage binding to this "AA" variant (SEQ. ID.NO. 24) was much weaker than to the wild type peptide (FIG. 1),indicating an important role for the two aspartate residues in theinteraction. The residual binding to the "AA" peptide (SEQ. ID. NO. 24)had the same cation requirements as the binding to the wild typepeptide.

B. Antibodies against CWDDGWLC Peptide Recognize β₃ and β₁ inImmunoblots

Further evidence for the structural similarity between CWDDGWLC (SEQ.ID. NO. 1) and β subunits was obtained with antibodies raised againstCWDDGWLC (SEQ. ID. NO. 1), whose preparation is described below. Whenpurified α_(IIb) β₃ was probed in immunoblots with anti-CWDDGWLC (SEQ.ID. NO 1), a band was detected that had the expected molecular size ofβ₃ and aligned with the band detected by anti-β₃ cytoplasmic domainantiserum. Reactivity of the anti-CWDDGWLC (SEQ. ID. NO 1) serum couldbe abrogated by preincubation of the serum with either CWDDGWLC (SEQ.ID. NO. 1) or β₃ (109-133) peptide (SEQ. ID. NO. 23). The anti-CWDDGWLC(SEQ. ID. NO 1) serum also reacted with bands that co-migrated with β₁and β₃ subunits in MG-63 total cell extracts. Anti-β₁ monoclonalantibody TS2/16 against β₁ was used as a positive control. Thereactivity of anti-CWDDGWLC (SEQ. ID. NO 1) serum was abrogated bypre-incubation of the antiserum with either CWDDGWLC (SEQ. ID. NO. 1) orβ₃ (109-133) (SEQ. ID. NO. 23). As expected, these peptides had noeffect on the reactivity of the positive control antibodies in a or b.

Immunization was performed as follows. RBF-Dnj mice (JacksonLaboratories, Bar Harbor, Me.) were immunized with 200 μg CWDDGWLC (SEQ.ID. NO. 1) peptide coupled to sheep red blood cells (Sigma) byintraperitoneal injection every 15 days for 3 months according to themanufacturer's instructions.

Immunoblot analysis of anti-CWDDGWLC (SEQ. ID. NO. 1) antiserum wasperformed as follows. Purified α_(IIb) β₃ (2 μg/lane) and MG-63 cellextracts (20 μl from a vol/vol detergent/cell pellet solution) wereseparated on 4-12% gradient SDS-PAGE and transferred to Immobilon-Pmembranes. After blocking of non-specific sites, filters were probedwith anti-CWDDGWLC (SEQ. ID. NO. 1) at a 1:500 dilution and anti-β₃cytoplasmic domain polyclonal serum at a 1:2,000 dilution. MG-63osteosarcoma cell extracts also were probed with TS2/16 (10 μg/ml),which is an anti-β₁ monoclonal antibody. Normal mouse and rabbit serawere used as negative controls. To confirm specificity of the serum,filters were incubated with anti-CWDDGWLC (SEQ. ID. NO. 1) serum in thepresence of 100 μM of the CWDDGWLC (SEQ. ID. NO. 1) or the β₃ (119-133)(SEQ. ID. NO. 23) peptides in solution.

Purified α_(IIb) β₃ was from Enzyme Research Laboratories Inc. (SouthBend, Ind.). Anti-β₁ monoclonal antibody TS2/16 was provided by Dr.Martin Hemler (Dana Farber Cancer Institute, Harvard Medical School,Boston, Mass.). Reactivity of antibodies was detected with anti-mouse orrabbit IgG, and chemiluminescence (ECL; Amersham).

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 28    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 8 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ    -      Cys Trp Asp Asp Gly Trp Leu Cys    #  5 1    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 9 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ    -      Cys Trp Asp Asp Leu Trp Trp Leu - # Cys    #  5 1    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 8 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ    -      Cys Trp Asp Asp Gly Leu Met Cys    #  5 1    - 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     Cys Thr Leu Arg Phe Gln Arg Ser - # Cys    #  5 1    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 9 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:8: (xi) SEQUENCE DESCRIPTION: SEQ    -      Cys Ser Trp Asp Asp Gly Trp Leu - # Cys    #  5 1    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 9 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:9: (xi) SEQUENCE DESCRIPTION: SEQ    -      Cys Pro Asp Asp Leu Trp Trp Leu - # Cys    #  5 1    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 8 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:10:(xi) SEQUENCE DESCRIPTION: SEQ    -      Cys Asp Gly Trp Leu Gly Phe Cys    #  5 1    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 10 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:11:(xi) SEQUENCE DESCRIPTION: SEQ    -      Cys Gln Arg Ile Val Leu Gly Phe - # Thr Cys    #   10    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 8 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:12:(xi) SEQUENCE DESCRIPTION: SEQ    -      Cys Asp Tyr Trp Leu Gly Phe Cys    #  5 1    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 8 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:13:(xi) SEQUENCE DESCRIPTION: SEQ    -      Cys Phe Val Leu Trp Leu Val Cys    #  5 1    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 7 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:14:(xi) SEQUENCE DESCRIPTION: SEQ    -      Cys Gly Asn Arg Leu Arg Cys    #  5 1    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 6 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    #ID NO:15:(xi) SEQUENCE DESCRIPTION: SEQ    -      Gly Arg Gly Asp Ser Pro    #  5 1    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 9 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:16:(xi) SEQUENCE DESCRIPTION: SEQ    -      Cys Glu Leu Arg Gly Asp Gly Trp - # Cys    #  5 1    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 10 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:17:(xi) SEQUENCE DESCRIPTION: SEQ    -      Cys Arg Asp Pro Arg Ala Gln Asp - # Leu Cys    #   10    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 10 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:18:(xi) SEQUENCE DESCRIPTION: SEQ    -      Ala Cys Arg Gly Asp Gly Trp Met - # Cys Gly    #   10    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 13 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    #ID NO:19:(xi) SEQUENCE DESCRIPTION: SEQ    -      Gly Ala Cys Val Arg Leu Asn Ser - # Leu Ala Cys Gly Ala    #   10    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 8 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1    #/note= "Xaa is an independently    #amino acid."  selected    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 2    #/note= "Xaa is an amino acid that    #with the amino acid at position 7 equals                   0 to 4 - # amino acids, each amino acid of which is                   independentl - #y selected."    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 5    #/note= "Xaa is an amino acidON:    #from the group consisting of glycine and                   leucine."    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 6    #/note= "Xaa is an amino acidON:    #from the group consisting of tryptophan                   and leuci - #ne."    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 7    #/note= "Xaa is an amino acid that    #with the amino acid at position 2 equals                   0 to 4 - # amino acids, each amino acid of which is                   independentl - #y selected."    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 8    #/note= "Xaa is an independently    #amino acid."  selected    #ID NO:20:(xi) SEQUENCE DESCRIPTION: SEQ    -      Xaa Xaa Asp Asp Xaa Xaa Xaa Xaa    #  5 1    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 6 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    #ID NO:21:(xi) SEQUENCE DESCRIPTION: SEQ    -      Gly Arg Gly Glu Ser Pro    #  5 1    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 10 amino              (B) TYPE: amino acid              (D) TOPOLOGY: circular    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 1    #/note= "Xaa is an independently    #amino acid."  selected    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 2    #/note= "Xaa is an amino acid that    #with the amino acid at position 9 equals                   0 to 3 - # amino acids, each amino acid of which is                   independentl - #y selected."    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 3    #/note= "Xaa is an amino acidON:    #from the group consisting of tryptophan                   and proli - #ne."    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 6    #/note= "Xaa is an amino acidON:    #from the group consisting of glycine and                   leucine."    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 7    #/note= "Xaa is an amino acidON:    #from the group consisting of tryptophan                   and leuci - #ne."    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 8    #/note= "Xaa is an amino acidON:    #from the group consisting of leucine,    #and methionine."yptophan    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 9    #/note= "Xaa is an amino acid that    #with the amino acid at position 2 equals                   0 to 3 - # amino acids, each amino acid of which is                   independentl - #y selected."    -     (ix) FEATURE:              (A) NAME/KEY: Peptide              (B) LOCATION: 10    #/note= "Xaa is an independently    #amino acid"   selected    #ID NO:22:(xi) SEQUENCE DESCRIPTION: SEQ    -      Xaa Xaa Xaa Asp Asp Xaa Xaa Xaa - # Xaa Xaa    #   10    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 25 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    #ID NO:23:(xi) SEQUENCE DESCRIPTION: SEQ    -      Asp Tyr Pro Val Asp Ile Tyr Tyr - # Leu Met Asp Leu Ser Tyr Ser    Met    #   15    -      Lys Asp Asp Leu Trp Ser Ile Gln - # Asn    #                 25    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 25 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    #ID NO:24:(xi) SEQUENCE DESCRIPTION: SEQ    -      Asp Tyr Pro Val Asp Ile Tyr Tyr - # Leu Met Asp Leu Ser Tyr Ser    Met    #   15    -      Lys Ala Ala Leu Trp Ser Ile Gln - # Asn    #                 25    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 28 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    #ID NO:25:(xi) SEQUENCE DESCRIPTION: SEQ    -      Asp Ile Tyr Tyr Leu Met Asp Leu - # Ser Tyr Ser Met Lys Asp Asp    Leu    #   15    -      Trp Ser Ile Gln Asn Leu Gly Thr - # Lys Leu Ala Thr    #                 25    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 28 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    #ID NO:26:(xi) SEQUENCE DESCRIPTION: SEQ    -      Asp Leu Tyr Tyr Leu Met Asp Leu - # Ser Tyr Ser Met Lys Asp Asp    Leu    #   15    -      Glu Asn Val Lys Ser Leu Gly Thr - # Asp Leu Met Asn    #                 25    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 29 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    #ID NO:27:(xi) SEQUENCE DESCRIPTION: SEQ    -      Asp Leu Tyr Tyr Leu Met Asp Leu - # Ser Leu Ser Met Lys Asp Asp    Leu    #   15    -      Asp Asn Ile Arg Ser Leu Gly Thr - # Lys Leu Ala Glu Glu    #                 25    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 29 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    #ID NO:28:(xi) SEQUENCE DESCRIPTION: SEQ    -      Asp Leu Tyr Tyr Leu Met Asp Leu - # Ser Ala Ala Met Asp Asp Asp    Leu    #   15    -      Asn Thr Ile Lys Glu Leu Gly Ser - # Gly Leu Ser Lys Glu    #                 25    __________________________________________________________________________

We claim:
 1. A compound having RGD-binding activity, comprising a cyclic peptide coupled to a moiety,wherein said cyclic peptide has the amino acid sequence:X₁ X₂ DDX₄ X₅ X₇ X₈ (SEQ. ID. NO 20),wherein X₁ and X₈ each is an independently selected amino acid; X₂ and X₇ together equal 0 to 4 amino acids, each amino acid of which is independently selected; X₄ is selected from the group consisting of glycine and leucine; and X₅ is selected from the group consisting of tryptophan and leucine and wherein said moiety is selected from the group consisting of a phage, polypeptide, carrier molecule, solid support and chemical molecule.
 2. The compound of claim 1, wherein X₁ and X₈ each is independently selected from the group consisting of cysteine; penicillamine; β,β-pentamethylene-β-mercaptopropionic acid; β,β-pentamethylene cysteine and functional equivalents thereof.
 3. The compound of claim 1, wherein X₁ and X₈ each is cysteine.
 4. The compound of claim 2, wherein X₄ is glycine and X₅ is tryptophan.
 5. The compound of claim 1, wherein said RGD-binding activity is divalent cation-independent.
 6. The compound of claim 1, wherein said moiety is a phage.
 7. The compound of claim 1, wherein said moiety is a polypeptide.
 8. The compound of claim 1, which is a fusion protein.
 9. A compound having RGD-binding activity, comprising a cyclic peptide coupled to a moiety, wherein said cyclic peptide has the amino acid sequence:X₁ X₂ X₃ DDX₄ X₅ X₆ X₇ X₈ (SEQ. ID. NO 22),wherein X₁ and X₈ each is an independently selected amino acid; X₂ and X₇ together equal 0 to 3 amino acids, each amino acid of which is independently selected; X₃ is selected from the group consisting of tryptophan and proline; X₄ is selected from the group consisting of glycine and leucine; X₅ is selected from the group consisting of tryptophan and leucine; and X₆ is selected from the group consisting of leucine, tryptophan and methionine and wherein said moiety is selected from the group consisting of a phage, polypeptide, carrier molecule, solid support and chemical molecule.
 10. The compound of claim 9, wherein X₁ and X₈ each is independently selected from the group consisting of cysteine; penicillamine; β,β-pentamethylene-β-mercaptopropionic acid; β,β-pentamethylene cysteine and functional equivalents thereof.
 11. The compound of claim 10, wherein X₁ and X₈ each is cysteine.
 12. The compound of claim 9, wherein said RGD-binding activity is divalent cation-independent.
 13. The compound of claim 9, wherein said moiety is a phage.
 14. The compound of claim 9, wherein said moiety is a polypeptide.
 15. The compound of claim 14, which is a fusion protein.
 16. The compound of claim 1, wherein said moiety is a carrier molecule.
 17. The compound of claim 1, wherein said moiety is a solid support.
 18. The compound of claim 1, wherein said moiety is a chemical molecule.
 19. The compound of claim 9, wherein said moiety is a carrier molecule.
 20. The compound of claim 9, wherein said moiety is a solid support.
 21. The compound of claim 9, wherein said moiety is a chemical molecule. 